• L Cipolla, A Consonni, G Guidetti, I Canobbio, M Okigaki, M Falasca, E Ciraolo, E Hirsch, C Balduini, and M Torti.
• Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.
• J. Thromb. Haemost. 2013 Feb 1; 11 (2): 345-56.

BackgroundThe proline-rich tyrosine kinase Pyk2 is a focal adhesion kinase expressed in blood platelets, and is activated downstream of G-protein coupled receptors as well as integrin α2β1.ObjectiveIn this study we have investigated the involvement of Pyk2 in integrin αIIbβ3 outside-in signaling in human and murine platelets.MethodsWe analyzed the stimulation of intracellular signaling pathways in platelets from Pyk2 knockout mice adherent to immobilized fibrinogen.ResultsPyk2 was rapidly phosphorylated and activated in human and murine platelets adherent to fibrinogen through integrin αIIbβ3. Activation of Pyk2 was Src-dependent, but did not require phospholipase Cγ2 activity. Platelets from Pyk2 knockout mice showed a defective ability to adhere and spread on fibrinogen, in association with a dramatic reduction of phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation. Pharmacological and genetic analysis demonstrated that integrin αIIbβ3 engagement selectively stimulated the β-isoform of PI3K (PI3Kβ), and that, as for Pyk2, PI3Kβ activation required Src family kinases activity, but not phospholipase Cγ2. In fibrinogen-adherent platelets, both Pyk2 and PI3Kβ were necessary for stimulation of the small GTPase Rap1b, a regulator of cell adhesion and spreading. Integrin αIIbβ3 engagement triggered the association of the PI3Kβ regulatory subunit p85 with the adaptor protein c-Cbl, which was mediated by the p85 SH3 domain, and was independent of c-Cbl tyrosine phosphorylation. However, p85-associated c-Cbl was tyrosine phosphorylated by activated Pyk2 in fibrinogen adherent platelets.ConclusionsThese results identify a novel pathway of integrin αIIbβ3 outside-in signaling and recognize the tyrosine kinase Pyk2 as a major regulator of platelet adhesion and spreading on fibrinogen.© 2012 International Society on Thrombosis and Haemostasis.

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